Compositions of chlorogenic acid and methods for making and using the same in obesity management

ABSTRACT

The present invention is directed compositions derived from green coffee bean extract and methods for the use and manufacture of such compositions. The compositions of the invention have unique ratios of chlorogenic acids which offer a therapeutic effect in the treatment of a variety of conditions and disorders. Methods for using the compositions of the invention include, but not limited to, methods for treating obesity and methods for regulating serum lipids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to provisionalapplication Ser. No. 62/031,135 filed Jul. 30, 2014, the contents ofwhich are incorporated herein by reference in their entirety for allpurposes.

FIELD OF THE INVENTION

The invention generally relates to compositions of chlorogenic acids andmethods for their manufacture and use in managing obesity. Moreparticularly, such compositions relate to formulations of chlorogenicacids from green coffee extract.

BACKGROUND

Obesity is a condition manifesting almost directly as a consequence ofmodern day's lifestyle that encompasses sedentary work-culture, highfat, calorie-rich diet, dearth of regular exercise or physical activity,addiction to habit forming substances such as tobacco and alcohol andhigh day-to-day stress levels. Obesity has reached epidemic proportionsglobally, with more than 1 billion adults overweight—at least 300million of them clinically obese—and is a major contributor to theglobal burden of chronic disease and disability. Ischemic heart diseaseor cardiovascular diseases are the conditions, often referred to aslifestyle diseases, have obesity as one of their root causes. Ischemicheart disease is the number one cause of death in the world today,according to a recent World Health Organization (WHO) report that may befound at http://who.int/mediacentre/factsheets/fs310/en/. Cardiovasculardiseases (CVDs) have killed nearly 17 million people in the year 2011which amounts to 3 in every 10 deaths. CVDs are among the top causes ofdeath, in India as well, as per the WHO. The importance of managingobesity is, thus, evident.

Often coexisting in developing countries with under-nutrition, obesityis a complex condition, with serious social and psychologicaldimensions, affecting virtually all ages and socioeconomic groups.Obesity and overweight pose a major risk for other serious chronicdiseases, including type 2 diabetes, hypertension, and stroke andcertain forms of cancer. The health consequences range from increasedrisk of premature death, to serious chronic conditions that reduce theoverall quality of life.

It is, therefore, safe to state that managing obesity wouldsubstantially aid in reducing the global mortality, increasing the lifeexpectancy and increasing the quality of life. Dietary changes, exerciseand activity, behavioral change, prescription weight-loss medicationsand weight-loss surgery are the common treatment arms for managingobesity. The treatment method to be undertaken often depends on thepreferred choice of an individual undergoing treatment as well as thelevel of obesity.

The preferred treatment modality for weight loss is dieting and physicalexercise. However, due to busy schedules and sedentary lifestyles,following-up with the first two methods seems to be practiced in anirregular manner. Weight loss surgery, on the other hand, is ruled outby a host of the population due to high costs involved. Therefore, thereis a gradual shift towards an increase in the use of drugs.

The drugs used for weight-loss, generally alter one of the fundamentalprocesses of the human body such as weight regulation by alteringappetite, metabolism or absorption of calories. Orlistat is the onlyanti-obesity medication which is currently approved by the FDA for longterm use. It reduces the intestinal fat absorption by inhibiting thepancreatic enzyme lipase. Rimonabant and Sibutramine are the other drugsthat had initially been approved for the treatment of obesity, but werebanned eventually due to safety concerns. Because of the potential sideeffects, it is recommended that anti-obesity drugs only be prescribedfor obesity where it is hoped that the benefits of the treatmentoutweigh its risk.

What is needed in the art therefore is a nutrition-based interventionthat provides an inexpensive alternative to aid weight loss and weightmanagement. The inventors of the present invention, therefore, envisagea cost-effective and safe herbal composition or a dietary supplementwhich is used for the management of obesity.

SUMMARY OF THE INVENTION

Some of the objects of the present disclosure are described hereinbelow:

It is an object of the present disclosure to provide a composition forthe management of obesity.

It is another object of the present disclosure to provide a compositionfor the management of obesity, which is cost-effective and non-toxic.

It is yet another object of the present disclosure to provide acomposition of CGA-7 isomers from Indian Green coffee bean extract forreducing body weight, removal of abdominal fat tissue, removal BAT,reducing cholesterol levels, improving HDL levels and restoring glucoselevels.

It is still another object of the present disclosure to provide aprocess for the preparation of CGA-7 Complex.

It is yet another object of the present disclosure to provide a methodfor the treatment and/or prevention of diseases associated with obesity,amongst others.

Other objects and advantages of the present disclosure will be moreapparent from the following description which is not intended to limitthe scope of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C show an MS chromatogram of a composition of the invention.

FIG. 2 shows an LS chromatogram of a composition of the invention.

FIG. 3 shows the percent reduction of body mass index resulting from theadministration of a composition of the invention.

FIG. 4 shows the percent reduction of body mass index resulting from theadministration of a composition of the invention.

FIG. 5 shows the percent reduction of waist circumference resulting fromthe administration of a composition of the invention.

FIG. 6 shows the percent reduction of hip circumference resulting fromthe administration of a composition of the invention.

FIG. 7 shows the effect of a composition of the invention on lipidprofile.

FIGS. 8A and 8B show that weight gain and average feed intake patternwhere CGA7 complex at 100 and 150 mg/kg show decrease in weight gaincompared to HFD and GCBE groups which is statistically significant.There is no difference in feed intake in between HFD and other treatedgroup.

FIGS. 9A-9E represent changes in liver and adipose tissues weight. CGA7complex at 100 and 150 mg·kg dose inhibits fat deposition in liver, BAT,mesenteric fat, epididymal and perirenal fat compared to HFD and GCBEgroups which is statistically significant.

FIGS. 10A-10D show changes in cholesterol, TG, glucose and HDL bloodserum level. CGA7 complex 100 and 150 mg·kg groups have decreasedcholesterol, TG and glucose compared to HFD and GCBE groups which isstatistically significant. CGA7 complex treated groups havesignificantly improved HDL level.

FIG. 11 shows the structure of ligand molecules: (1) 3-O-Caffeoylquinicacid; (2) 4-O-Caffeoylquinic acid; (3) 5-O-Caffeoylquinic acid; (4)5-O-Feruloylquinic acid; (5) 3,4-O-Dicaffeoylquinic acid; (6)3,5-O-Dicaffeoylquinic acid; (7) 4,5-O-Dicaffeoylquinic acid; and (8)Orlistat.

DETAILED DESCRIPTION

In accordance with one aspect of the present disclosure, there isprovided a process for the preparation and use of a compositioncomprising one or more chlorogenic acids (e.g. a CGA-7 complex). Thepart plants used for the preparation of the composition of the inventionmay be fresh and/or dried. Typically, the composition is prepared byobtaining an extract of one or more chlorogenic acids. Such extract canbe obtained by processes including, but not limited to, alcoholic,hydro-alcoholic and aqueous extract. Further, the composition isprepared by using techniques selected from the group that includes butis not limited to hot stirring, Soxhlet extraction, percolation,decoction, maceration and supercritical fluid extraction. The solventused for extraction can be a polar organic solvent and/or a non-polarorganic solvent. Solvents suitable for use with the invention include,but are not limited to water, methanol, ethanol, butanol, hexane,acetone, chloroform, petroleum ether and acetonitrile. In one embodimentof the present disclosure, green coffee beans are subjected toextraction to obtain a combination of one or more chlorogenic acids.

The composition may be formulated to attain a particular chlorogenicacid content. The composition may have a total chlorogenic acid contentof between about 20-60% w/w. The composition may have a totalchlorogenic acid content, by weight, of about 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, as well as any amountintervening these specifically described amounts. The total chlorogenicacid content may be 42.50±2.5 w/w %. The total chlorogenic acid contentmay be constituted from one or more of 3 CQA, 5 CQA, 4 CQA, 3,4 Di CQA,3,5 Di CQA, 4,5 Di CQA. The total chlorogenic acid content may contain,for example, a chlorogenic acid content of about 1-15% w/w 3 CQA, about5-50% w/w 5 CQA, about 1-20% w/w 4 CQA, about 0.5-10% w/w 3,4 Di CQA,about 0.5-10% w/w 3,5 Di CQA, about 0.5-10% w/w 4,5 Di CQA, orcombination thereof. The composition may contain 4.1±1.42 w/w % 3CQA,28±4.65 w/w % 5 CQA, 6.5±2.25 w/w % 4 CQA, 0.84±0.26 w/w % 3,4 Di CQA,1.23±0.34 w/w % 3,5 Di CQA, and 1.85±0.42 w/w % 4,5 Di CQA.

In one embodiment of the present invention, the composition comprises acombination of chlorogenic acids. In one non-limiting embodiment, thecomposition comprises 3-caffeoylquinic acid (3-CQA), 5-caffeoylquinicacid (5-CQA), 4-caffeoylquinic acid (4-CQA), 5-feruloylquinic acids(5-FQA), 3,4 dicaffeoylquinic acids (3,4 di CQA), 3,5 dicaffeoylquinicacids (3,5 di CQA), and 4,5 dicaffeoylquinic acids (4,5 di CQA).

In one non-limiting embodiment, the composition comprises3-caffeoylquinic acid (3-CQA) at about 7.23%, 5-caffeoylquinic acid(5-CQA) at about 25.43%, 4-caffeoylquinic acid (4-CQA) at about 9.51%,5-feruloylquinic acids (5-FQA) at about 1.84%, 3,4 dicaffeoylquinicacids (3,4 di CQA) at about 2.79%, 3,5 dicaffeoylquinic acids (3,5 diCQA) at about 1.90%, and 4,5 dicaffeoylquinic acids (4,5 di CQA) atabout 3.74%.

The term “about” as used herein refers to a quantity, level, value,number, frequency, percentage, dimension, size, amount, weight or lengththat varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or1% to a reference quantity, level, value, number, frequency, percentage,dimension, size, amount, weight or length. In particular embodiments,the terms “about” or “approximately” when preceding a numerical valueindicates the value plus or minus a range of 15%, 10%, 5%, or 1%, or anyintervening range thereof.

In accordance with another aspect of the present disclosure, there isprovided a process for the preparation of a dosage form comprising thecomposition of the invention. In accordance with yet another aspect ofthe present disclosure there is provided a complex mixture for managingobesity that comprises a combination of chlorogenic acids. Thephyto-constituents included in the composition of the present disclosurecan be in any form that permits an effective dosage for use such as themanagement of obesity. Suitable forms for administering the compositioninclude, but are not limited to, extracts, granules, powders,semisolids, isolated fractions, oils, solutions, suspensions, emulsionsand semisolids. In one embodiment, the composition is a complex mixtureobtained from Indian Green coffee bean.

The composition of the present invention may be administered by variousroutes including but not limiting to topical, oral, buccal, sub-lingual,parenteral, rectal, and inhalation. The composition may be in the formof a dosage form that includes but is not limited to powders, pills,tablets, pellets, capsules, thin films, solutions, sprays, syrups,linctuses, lozenges, pastilles, chewing gums, pastes, vaporizers,suspensions, emulsions, ointments, creams, lotions, liniments, gels,drops, topical patches, buccal patches, injections and the like.Typically, the composition further comprises at least onepharmaceutically acceptable excipient.

The composition of the present invention can be used in any applicationin which administration of the composition to a subject providesbeneficial health effects. Suitable applications for administration ofthe composition include, but are not limited to, preventing oxidation(antioxidant activity), controlling and/or reducing body weight,controlling and/or reducing body mass index, controlling and/or reducingobesity, management of hyperlipidemic conditions, reducing oxidativestress, maintaining a healthy lipid profile, regulating blood glucoselevels, treatment of liver cirrhosis, treatment of atherosclerosis, andtreatment bacterial infection. The composition of the invention may beadministered to reduce low density lipids. The composition of theinvention may be administered to increase high density lipids. Thecomposition of the invention may be administered to reduce low densitylipids and increase high density lipids. Significantly, the compositionis non-toxic and non-mutagenic. In some aspects of the The phrase “bodymass index” as used herein refers to a ratio of height to body weightthat is calculated as follows:

${BMI} = {\frac{{mass}_{kg}}{{height}_{m}^{2}} = {\frac{{mass}_{lb}}{{height}_{in}^{2}} \times 703}}$

The terms “obese” and “obesity” as used herein refer to a subject havinga body mass index of 30 or higher.

The term “overweight” as used herein refers to a subject having a bodymass index of 25 to 29.9.

The term “healthy weight” as used herein refers to a subject having abody mass index of 18.5 to 24.9.

The phrase “treating obesity” as used herein refers to reducing BMI inan obese subject, reducing body fat in an obese subject, or reducingbody weight in a subject.

As used herein, the term “controlling” refers to maintaining the stateof a condition so as to prevent an increase or decrease in the conditionor an increase or decrease in the symptoms of the condition. Controllingmay refer to controlling obesity wherein the state of obesity of asubject is maintained such that the body weight or body mass index of asubject is maintained at a level without any appreciable increase inbody weight or body mass index.

As used herein, the term “reduce,” “reducing” and the like refer to anymeasurable decrease in a condition or parameter that results from theadministration of the composition of the invention to a subject whencompared to the absence of the administration of the composition, suchas compared to the administration of a control composition (e.g. aplacebo). In some aspects of the invention, administration of acomposition of the invention to a subject reduces obesity or body weightin the subject when compared to the administration of a controlsubstance to a subject. In some aspects of the invention, administeringthe compositions of the invention maintains body weight (i.e. preventsan increase in body weight or an increase in percent body fat).

As used herein, the terms “treatment,” “treating,” and the like, whenused in reference to a subject, refer to obtaining a desiredpharmacologic and/or physiologic effect, including without limitationachieving a reversal, improvement or elimination of the symptoms of adisease or condition. The effect can be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/orcan be therapeutic in terms of achieving an improvement or eliminationof symptoms, or providing a partial or complete cure for a diseaseand/or adverse affect attributable to the disease. “Treatment,” as usedherein, covers any treatment of a disease in a mammal, particularly in ahuman, and includes: (a) preventing the disease from occurring in asubject which can be predisposed to the disease but has not yet beendiagnosed as having it; (b) inhibiting the disease, i.e., arresting itsdevelopment; (c) relieving the disease, e.g., causing regression of thedisease, e.g., to completely or partially eliminate symptoms of thedisease; and (d) restoring the individual to a pre-disease state, e.g.,reconstituting the hematopoietic system.

The compositions of the invention can be administered to treat obesityin a subject. The compositions of the invention can be administered toreduce body mass index in an obese subject. The compositions of theinvention can be administered to reduce body mass index in an overweightsubject. The compositions of the invention may be administered tocontrol or prevent weight gain in a subject. In some aspects of theinvention may be administered to maintain or achieve a healthy weight ina subject. The compositions of the invention can be administered toreduce body fat in an obese subject. The compositions of the inventioncan be administered to reduce body fat in an overweight subject. Thecompositions of the invention may be administered to control or preventbody fat gain in an obese subject. The compositions of the invention maybe administered to control or prevent body fat gain in an overweightsubject. The compositions of the invention may be administered tocontrol or prevent body fat gain in a healthy weigh subject.

As used herein, “administer” or “administration” refers to the act ofphysically delivering a substance as it exists outside the body into asubject. Administration includes all forms known in the art fordelivering therapeutic agents, including but not limited to oral,topical, mucosal, injections, intradermal, intravenous, intramusculardelivery or other method of physical delivery described herein or knownin the art (e.g., implantation of a slow-release device, such as amini-osmotic pump to a subject; liposomal formulations; buccal;sublingual; palatal; gingival; nasal; vaginal; rectal; intra-arteriole;intraperitoneal; intraventricular; intracranial; or transdermal). Thecompositions described herein can be administered intravenously.

The compositions of the invention may be administered in an effectiveamount. The phrase an “effective amount” as used herein refers to anamount of a composition of the invention that is sufficient to achievethe effect for which the composition is administered (e.g., to maintainor reduce obesity).

The terms “subject,” “patient,” “subject in need thereof,” and “patientin need thereof” are herein used interchangeably and refer to mammal,including humans. Subjects may also be cattle, sheep, goats, pigs orother animals used in animal husbandry. In a preferred embodiment thesubject is a human. The subject can be a human suffering from obesity orhaving an unhealthy body weight.

The composition of the invention may be obtained from any sourcematerial capable of providing the chlorogenic acids disclosed herein.The compositions of the invention can be obtained from green coffee beanmaterial. In one non-limiting embodiment of the invention, thecomposition is derived from Indian green coffee bean material. The term“green coffee bean” as used herein refers to unroasted mature orimmature coffee beans. Green coffee beans can be processed by wet or drymethods for removing the outer pulp and mucilage, and can have an intactwax layer on the outer surface. When immature coffee beans are immature,they are green. When mature, they have a brown to yellow or reddishcolor, and typically weigh 300 to 330 mg per dried coffee bean. Thecompositions of the invention may comprise green coffee extract as anextract of unroasted, green coffee beans. The compositions of theinvention may be obtained from the green coffee bean. The compositionsof the invention may be obtained from the outer pulp (mucilage) of thebean. The compositions of the invention may be obtained from the greencoffee bean. The compositions of the invention may be obtained from acombination of the green coffee bean and the pulp of the coffee bean.

The present disclosure is further described in the light of thefollowing non-limiting examples which are set forth for illustrationpurpose only and not to be construed for limiting the scope of thepresent invention.

Example 1 Process for Preparation of the Complex Mixture of the PresentDisclosure (CGA-7 Complex)

100 kg of Green coffee bean was stacked in a vertical 1.0 KL extractor.The bottom of the extractor comprised of a perforated plate on whichfiltration cloth was fixed. About six bed volumes of 70% v/v ethylalcohol was added. Extraction was continued at 75-78° C. about 7-8 hrswith continuous circulation of extract with transfer pump. Aftercompletion of extraction, filter the extract through 5 micron SS candlefilter and clear extract was collected in a cleaned receiver tank. Thebed was re-extracted by adding 4 bed volumes of 70% ethyl alcohol 3 moretimes and temperature was maintained at 75-78° C. about 7-8 hrs. All theextracts were collected in a receiver tank and combined extract wasconcentrated in a reactor under vacuum at 80±5° C. till extract was freefrom ethyl alcohol. Solution was made up to the TDS to 20-25 w/v % withde-ionized water.

The above extract solution was passed through 500 Liter of XAD-4 resinand the extract was loaded through the resin at the rate of 2-3 bedvolumes/hour. The resin was washed with 2-3 bed volumes of de-ionizedwater at the rate of 2-3 bed volumes/hour. The extract was eluted with2-3 bed volumes of 70 v/v % Ethyl alcohol at the rate of 2-3 bedvolumes/hour. The eluent was concentrated in a reactor at 80±5° C. tillfree from alcohol. The solution TDS was made up to 25-30 w/v % and spraydried at 215±5° C. to obtain the complex mixture of CGA-7. Thecomposition of the chlorogenic acid isomers was determined by the HPLCmethod and the following results were obtained:

TABLE 1 Peak Table showing Types of Chrologenic acids Ret. Peak NameTime Area Area % Height Height % 1 3 CQA 3.209 1904533 12.529 39588013.745 2 5 CQA 5.017 7126683 46.883 1394953 48.431 3 4 CQA 5.218 261257817.187 521622 18.110 4 5 FQA 7.489 703664 4.629 136587 4.742 5 3,4 Di11.049 941037 6.191 162285 5.634 CQA 6 3,5 Di 11.549 694548 4.569 1111033.857 CQA 7 4,5 Di 12.659 1218027 8.013 157843 5.480 CQA Total 15201070100 2880273 100

The fingerprint of the composition of the chlorogenic acid isomers wasdetermined by the LC-MS/MS method and the results that were obtained aredepicted in FIGS. 1A-C and FIG. 2.

Example 2 In-Vitro Studies of the Complex Mixture (CGA-7 Complex) of thePresent Disclosure Antioxidant Assay: DPPH Scavenging Assay

The free radical scavenging capacity of CGA-7 Complex was determinedusing DPPH scavenging assay (Sarojini et al., 2011). DPPH solution wasprepared in 95% methanol. Freshly prepared DPPH solution was taken intest tubes and coffee preparations were added and incubated for 20 min.The absorbance was read at 515 nm using a spectrophotometer. Blank wasprepared containing the same volume of reaction mixture without anytested samples. The percentage of scavenging was calculated usingformula

% Scavenging=Ac−As/Ac×100

Where AC was the absorbance of the control (blank without extract) andAs was the absorbance in the presence of the extract

Metal Chelating Activity

The chelating of ferrous ions by the Plant extracts was estimated by thefollowing method. CGA-7 Complex was added to a solution of 2 mM FeCl2(0.05 ml), the reaction was initiated by the addition of 5 mM ferrozine(0.2 ml). Then the mixture was shaken vigorously and kept for 10 min atroom temperature [26]. Absorbance of the solution was measured at 562nm. The percentage of inhibition of ferrozine-Fe2+ complex formation wascalculated.

Chelating rate (%)=Ac−As/Ac×100

Where AC was the absorbance of the control (blank without extract) andAs was the absorbance in the presence of the extract

Superoxide Anion Scavenging Activity

Superoxide anion scavenging activity of CGA-7 Complex was measuredaccording to the method of (Nishimiki et al., 1972). Prepare all thesolutions in this experiment using phosphate buffer (pH 7.4). Add 1 mlof NBT (156 μM), 1 ml of NADH (468 μM) and 3 ml of test samples. to alltest tubes. The reaction is started by adding 100 ml of PMS (60 μM) andincubate the mixture at 25° C. for 5 min followed by measurement ofabsorbance at 560 nm. The percentage of scavenging was calculated usingformula

% Scavenging=Ac−As/Ac×100

Where AC was the absorbance of the control (blank without extract) andAs was the absorbance in the presence of the extract

Reducing Power Assay

The reductive ability of CGA-7 Complex was determined. The test sampleswere mixed with 2.5 ml of 0.2 M phosphate buffer (pH 6.6) and 2.5 ml of1% potassium ferricyanide [K3Fe(CN)6]. Reaction mixture was incubated at50° C. for 20 min, add 2.5 ml of 10% trichloroacetic acid, thencentrifuge (650 rpm at room temperature) for 10 min. The upper layersolution (2.5 ml) was mixed with 2.5 ml of distilled water and 0.5 ml of0.1% FeCl3. Absorbance was measured at 700 nm. Higher the absorbance at700 nm indicates higher reducing power ability (Oyaizu, 1986).

Total Antioxidant Activity

The phosphomolybdenum method is based on the reduction of Mo (VI) to Mo(V) by the antioxidant compound and the formation of a greenphosphate/Mo (V) complex with a maximal absorption at 695 nm. Theantioxidant activity of the test sample will be determined by thephosphomolybdenum method as described by Prieto et al., (1999). Briefly,0.3 ml of test sample combined with 3 ml of reagent solution (0.6Msulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). Thereaction mixture will be incubated at 95° C. for 90 min and cooled toroom temperature. Measure the absorbance of the solution at 695 nmagainst blank. The total antioxidant capacity expressed as the number ofequivalents of ascorbic acid (AAE).

Results: Sample Name: CGA-7 Complex

TABLE 2 In-vitro assays of CGA-7 Complex DPPH Superoxide scavengingscavenging assay assay Metal chelating Conc Conc Conc in Absorbance % inAbsorbance % in Absorbance % μg/ml @ 517 nm inhibition μg/ml @ 560 nminhibition μg/ml @ 562 nm inhibition Blank 1.801 Blank 0.337 Blank 0.702100 μg 0.886 50.81 100 μg 0.110 67.36 100 μg 0.726 −3.42 1.007 44.090.102 69.73 0.739 −5.27 0.985 45.31 0.120 64.39 0.746 −6.27 0.967 46.310.123 63.50 0.734 −4.56 0.976 45.81 45.97 66.25 −4.88 Total Reducingantioxidant assay power assay Conc Conc in Absorbance AAE in AbsorbanceAAE μg/ml @ 695 nm (μg) μg/ml @ 700 nm (μg) 100 μg 0.078 14.78 100 μg0.545 58.55 0.078 14.80 0.590 63.37 0.080 14.95 0.598 64.16 0.074 14.500.560 60.09 0.078 14.80 0.555 59.63 14.76 61.16

Example 3 In-Vivo Studies of CGA-7 Complex of the Present Disclosure

A] Acute Oral Toxicity Study in Rats with CGA-7 Complex

Single-dose oral toxicity of the CGA-7 Complex was evaluated in albinoWistar rats. A limit test was performed in which female rats received asingle oral administration of the CGA-7 Complex at a dose of 2000 mg/kgbody weight. Following dosing, the limit test rats were observed dailyand weighed weekly. A gross necropsy examination was performed on alllimit test animals at the time of scheduled euthanasia (day 14). Nomortality occurred during the duration of the limit test. Further, nosignificant gross internal findings were observed at necropsy on studyday 14.

Under the experimental conditions described, the acute oral LD₅₀ ofCGA-7 complex was estimated to be greater than 2000 mg/kg in the rat.

B] Repeated Dose 90 Days Toxicity with CGA-7 Complex

Repeated dose 90 day oral toxicity study was performed with CGA-7complex in both male and female Wistar rats followed by 14 day recoveryperiod. To determine target organ toxicity, No observed adverse effectlevel (NOAEL) and reversibility of signs of toxicity after recoveryperiod.

Results of acute oral toxicity studies in Wistar albino rats indicatedthe acute oral LD₅₀ of CGA-7 complex was estimated to be greater than2000 mg/kg in the rats. Based on the above results; three doses wereselected as 250, 500 and 1000 mg/kg b.w. Oral route of administrationwas selected because it is the proposed therapeutic route.

Observations

Clinical signs of toxicity: No clinical signs of toxicity were observedin all male and female groups of animals throughout the dosing period of90 days and during the recovery period of 14 days.

Mortality: All the male and female groups of animals were survivedthroughout the dosing period of 90 days and during the recovery periodof 14 days.

Body Weight: Body weights were recorded 0 to 90 days for all groups andcontinued for day 97 and 104 for reversal groups.

Body weight gain: A significant decrease in body weight gain was seen inall groups of animals treated with CGA-7 Complex at 250, 500 and 1000mg/kg b.w. in both males and females when compared with control. Duringthe reversal period the animals also showed a significant decrease inbody weight gain to normal when compared with control reversal group.

Food Intake: No effect of treatment in all groups of animals was notedon food consumption throughout the dosing period of 90 days and duringthe recovery period of 14 days.

Functional observations: The functional observational parametersperformed showed no changes in all groups of male and female animals.

Blood Analysis

Hematology: No significant changes were observed in all groups of maleand female animals when compared with those of respective controlgroups. No dose dependent changes were seen.

Clinical Chemistry: No significant changes were observed in all groupsof males and females when compared with those of respective controlgroups.

Urine analysis: No changes were noted in Urine analysis of all groups ofmale and female animals when compared with those of respective controlgroups.

Sacrifice and Pathology

Relative Organ weight: No significant changes were observed in allgroups of male and female animals when compared with those of respectivecontrol groups. No dose dependent changes were observed.

Gross pathology: No gross changes were observed in all groups of maleand female animals.

Histopathological studies: No changes were seen in brain, liver, heart,spleen, kidneys, ovaries, large intestine and adrenal glands in animalstreated with CGA-7 Complex 1000 mg/kg b.w. when compared with control.

Conclusion: Based on the above findings, the no observed adverse effectlevel (NOAEL) of CGA-7 complex was found to be 1000 mg/kg·b.w. for bothfemale and male Wistar rats when given orally for 90 days followed by 14day recovery period.

C] Anti-Obesity Activity of the CGA-7 Complex:

Objective: The objective was to investigate the effect of the CGA-7complex against high fat diet fed rats.

Procedure:

Model: High fat diet in rats

Composition of High Fat Diet 25% Lard 5% Soyabean oil

5% starch65% Normal commercial available rat feed

Male Wistar rats were divided into six groups with six animals in eachgroup.

TABLE 3 Specifications of groups S. NO Group Test substance 1 Group INormal feed + Vehicle (distilled water) 2 Group II High fat diet +Vehicle (distilled water) 3 Group III High fat diet + Standard drug(orlistat 30 mg/kg) 4 Group IV High fat diet + CGA 7 Complex (50 mg/kg)5 Group V High fat diet + CGA 7 Complex (100 mg/kg) 6 Group VI High fatdiet + CGA 7 Complex (150 mg/kg)

Parameters Evaluated

Body weight was measured once every 2 days. Food intake was measureddaily for 42 days.

On day 43, all the animals were kept overnight fasting before sacrifice.Blood was collected by puncturing the retro orbital plexus. Serum wasseparated by centrifugation at 3000 rpm for 10 minutes.

Serum was estimated for glucose, cholesterol, triglycerides and HDL. LDLand atherogenic index were also calculated. Liver, mesenteric, brownadipose tissue (BAT), left and right perirenal fat pads, left and rightepididymal pads were isolated and weighed. Liver was isolated andestimated for liver triglycerides and cholesterol levels.

Statistical Analysis

Data were expressed as mean±SEM & analyzed by one way ANOVA followed byDunnett's t test using graph pad prism version 5. Differences wereconsidered significant at a p value of <0.05.

Results

TABLE 4 Effect of CGA 7 Complex on body weight in male rats Groups Finalweight Initial weight Difference in body weight Group I 220.5 ± 2.54150.1 ± 2.67 68.41 ± 1.18    Group II 230.3 ± 5.57 137.0 ± 0.32 112.0 ±4.51*** ^(a) Group III 218.4 ± 2.65 130.3 ± 0.56 88.01 ± 3.68*** ^(b)Group IV 211.7 ± 2.42 136.1 ± 0.62 75.65 ± 3.44*** ^(b) Group V 229.7 ±1.94 163.8 ± 2.33 65.88 ± 2.14*** ^(b) Group VI 199.0 ± 2.08 138.4 ±1.07 60.63 ± 2.89*** ^(b) Values are expressed in terms of SEM ± Mean.Data were analyzed by one way ANOVA followed by Dunnett's t test. Numberof animals in each group n = 6. ^(a) Comparison made with control group.^(b) Comparison made with high fat diet group. ***P < 0.001.

TABLE 5 Effect of CGA 7 Complex on average food intake in male ratsGroups Average food intake g/rat Group I 14.93 ± 0.77    Group II  8.26± 0.51***^(a) Group III 8.54 ± 0.40^(nsb) Group IV 7.78 ± 0.45^(nsb)Group V 8.96 ± 0.45^(nsb) Group VI 6.98 ± 0.35^(nsb) Values areexpressed in terms of SEM ± Mean. Data were analyzed by one way ANOVAfollowed by Dunnett's t test. Number of animals in each group n = 6.^(a)Comparison made with control group. ^(b)Comparison made with highfat diet group. ***P < 0.001 ^(ns)non significant.

TABLE 6 Effect of CGA 7 Complex on liver organ weight, mesenteric, brownadipose tissue (BAT), perirenal fat pads and epididymal fat pad.Mesenteric Peri renal fat pad tissue Epididymal fat pad Groups Liver fatBAT Right Left Right Left Group I 3.87 ± 0.18    0.59 ± 0.06   0.06 ±0.004   0.30 ± 0.02    0.28 ± 0.01    0.35 ± 0.03    0.38 ± 0.01   Group II 4.50 ± 0.19*^(a)   1.31 ± 0.12***^(a) 0.24 ± 0.01***^(a) 1.22 ±0.12***^(a) 0.85 ± 0.05***^(a) 0.58 ± 0.04***^(a) 0.60 ± 0.02***^(a)Group III 3.47 ± 0.11***^(b) 0.98 ± 0.03*^(b)  0.19 ± 0.01**^(b)  0.65 ±0.04*^(b)  0.61 ± 0.05*^(b)  0.48 ± 0.01*^(b)  0.50 ± 0.01**^(b)  GroupIV 3.56 ± 0.10***^(b) 1.01 ± 0.07*^(b)  0.20 ± 0.01*^(b)  0.52 ±0.03***^(b) 0.54 ± 0.03***^(b) 0.44 ± 0.02**^(b)  0.46 ± 0.03***^(b)Group V 3.57 ± 0.12***^(b) 1.0 ± 0.04*^(b) 0.17 ± 0.01***^(b) 0.43 ±0.02***^(b) 0.41 ± 0.03***^(b) 0.41 ± 0.02***^(b) 0.38 ± 0.02***^(b)Group VI 3.39 ± 0.10***^(b) 1.0 ± 0.07*^(b) 0.17 ± 0.01***^(b) 0.36 ±0.02***^(b) 0.38 ± 0.03**^(b)  0.40 ± 0.03***^(b) 0.37 ± 0.01***^(b)Data were analyzed by one way ANOVA followed by Dunnett's t test. Numberof animals in each group n = 6. ^(a)Comparison made with control group.^(b)Comparison made with high fat diet group. ***P < 0.001 **P < 0.01 *P< 0.05.

TABLE 7 Effect of CGA 7 Complex on serum glucose, total cholesterol,triglyceride, HDL-c and LDL-c levels (mg/dL) Total Groups Glucosecholesterol Triglyceride HDL-c LDL-c Group I 84.53 ± 4.62    56.00 ±1.44    70.56 ± 3.59    24.56 ± 0.88   17.33 ± 1.78   Group II 131.00 ±5.88***^(a)  78.08 ± 1.40***^(a) 137.7 ± 7.95***^(a)  16.73 ±0.59***^(a)  33.81 ± 2.11***^(a) Group III 109.7 ± 3.93**^(b)  63.38 ±0.95***^(b) 77.63 ± 4.27***^(b) 20.47 ± 1.08*^(b) 27.39 ± 1.07^(nsb) Group IV 89.29 ± 2.79***^(b) 62.23 ± 1.74***^(b) 86.32 ± 5.06***^(b)20.22 ± 0.82*^(b) 25.12 ± 2.33*^(b)  Group V 87.63 ± 3.28***^(b) 58.16 ±2.18***^(b) 75.36 ± 4.17***^(b) 20.50 ± 1.10*^(b) 22.59 ± 2.67**^(b)Group VI 86.23 ± 3.50***^(b) 45.20 ± 2.37***^(b) 62.98 ± 2.97***^(b)20.51 ± 0.94*^(b)  12.09 ± 2.31***^(b) Data were analyzed by one wayANOVA followed by Dunnett's t test. Number of animals in each group n =6. ^(a)Comparison made with control group. ^(b)Comparison made with highfat diet group. ***P < 0.001 **P < 0.01 *P < 0.05

TABLE 8 Effect of CGA 7 Complex on atherogenic index Group AtherogenicIndex Group I 1.30 ± 0.10    Group II 3.72 ± 0.22***^(a) Group III 2.20± 0.20***^(b) Group IV 2.18 ± 0.15***^(b) Group V 1.91 ± 0.23***^(b)Group VI 1.22 ± 0.14***^(b) Data were analyzed by one way ANOVA followedby Dunnett's t test. Number of animals in each group n = 6.^(a)Comparison made with control group. ^(b)Comparison made with highfat diet group. ***P < 0.001

TABLE 9 Effect of CGA 7 Complex on liver cholesterol & triglyceridelevels Group liver cholesterol liver triglycerides Group I 23.92 ±1.50    50.51 ± 2.16    Group II 69.53 ± 2.54***^(a) 87.93 ± 3.56***^(a)Group III 49.66 ± 3.28***^(b) 64.64 ± 3.44***^(b) Group IV 48.48 ±3.58***^(b) 57.68 ± 4.25***^(b) Group V 44.61 ± 4.45***^(b) 59.91 ±2.73***^(b) Group VI 28.53 ± 2.69***^(b) 36.04 ± 4.42***^(b) Data wereanalyzed by one way ANOVA followed by Dunnett's t test. Number ofanimals in each group n = 6. ^(a)Comparison made with control group.^(b)Comparison made with high fat diet group. ***P < 0.001 **P < 0.01 *P < 0.05

Conclusion

The CGA 7 complex reduces body weight, removes abdominal fat tissue,removes brown adipose tissue, reduces cholesterol levels, improves HDL clevels, and restores glucose levels.

Example 4 Clinical Evaluation of CGA-7 Complex of the Present DisclosureA Prospective, Randomized, Double Blind, Placebo Controlled ClinicalTrial to Evaluate Efficacy and Safety of CGA-7 Complex in Reducing theBodyweight in Obese Patients.

The subjects had to complete 5 visits including 1 screening visit and 4scheduled visits; i.e., screening visit, Visit 1 (baseline)—Day 1, Visit2—Day 14±3, Visit 3—Day 28±3, Visit 4—Day 56±3. The screening visitconsisted of patient consent form, multiple laboratory tests, physicalexaminations, anthropometric measurements and understandsinclusion/exclusion criteria. After screening, subjects were randomizedand assigned to Group A and Group B for CGA-7 Complex and placebotreatment respectively. Primary outcomes reduction in body weight andBMI on visit 2, visit 3 and visit 4 were recorded. Baseline quality oflife assessment (SF-36) was also carried out. During visit 2 & 3 theanthropometric measurements, physical examination, adverse events, andconcomitant medications if any were recorded. In final visit, physicalexaminations, anthropometric measurements, lipid profile, fasting bloodglucose, HbA1c, quality of life assessment, adverse events, concomitantmedications were recorded. The unused tablets were collected back.

The effect of CGA-7 Complex was validated in the absence of exercise,walking and without diet restrictions during the course of the trial.

Sample Size

Total 42 patients were screened and among them 30 were included in thestudy as per inclusion criteria. Subjects were randomized and equal noof subjects were assigned in CGA-7 Complex treatment group A and placebotreatment group B. Two patients were dropped out from both groupswithout any information during study.

Statistical Analysis

Paired t-test was used to measure the change from the baseline. ANOVAwas used in order to observe the changes in the various parameters afterthe follow up visits scheduled, followed by appropriate post-hoc test.

Results Body Weight

As shown in FIG. 3, in the CGA-7 Complex treated group, baseline meanbody weight was 80.6±10 which was reduced up to 76.57±10.2 on 56^(th)day. Where as in placebo treated group, on 56^(th) day weight reductionwas negligible compare to baseline mean body weight.

There was 5% reduction in body weight observed in CGA-7 Complex treatedgroup compared to baseline, which is significant compared to placebotreated group. In placebo treated group only 0.9% of reduction was seenin body weight compared to baseline.

BMI

As shown in FIG. 4, mean BMI of the CGA-7 Complex treated group atbaseline was 30.53±0.62 which was significantly reduced to 28.97±0.73 onlast visit. The same was found to be slightly decrease in placebotreated subjects.

CGA-7 Complex treated group has shown 5.10% reduction in BMI on 56^(th)day where as it was only 0.9% in placebo treated group.

Waist Circumference

As shown in FIG. 5, there was a significant reduction in waistcircumference on 56^(th) day in CGA-7 Complex treated group compared tobaseline which was 103.28±4.14 and 99.85±5.36, respectively. Whereas,placebo treated group had shown negligible improvement in waistcircumference.

Hip Circumference

As shown in FIG. 6, hip circumference was significantly improved in theCGA-7 Complex treated group compared to placebo treated group. Thebaseline mean hip circumference was 105.85±4.88 which was reduced to104.78±4.88 after 56 days treatment with CGA-7 Complex. 1.01% ofreduction has been seen in CGA-7 Complex treated group. Which was only0.13% in Placebo treated group.

Lipid Profile

As shown in FIG. 7, the results collected have shown that CGA 7 Complexhas significant improvement in lipid profile. The mean triglyceridelevel at baseline was 151±39.39 which decreased significantly to134.43±38.15 at Day 56 after treatment with CGA 7 Complex and thepercentage of triglyceride levels reduced compared to baseline is10.97%.

The mean total cholesterol values at baseline 172.07±32.99 whichdecreased to 162.36±32.10 at the Day 56 after treatment with CGA 7Complex and the Percentage of Total cholesterol levels reduced comparedfrom baseline to visit 4 is 5.64%.

The mean HDL level at baseline was 37.91±8.29 which increasedsignificantly to 42±7.75 at Day 56 after treatment with CGA 7 Complexand the Percentage of HDL level increased is 10.78%.

The mean LDL level at baseline was 103.96±27.18 which decreasedsignificantly to 88.5±29.62 at Day 56 after treatment with CGA 7 Complexand the Percentage of LDL levels reduced compared from baseline to visit4 is 14.87%

The mean VLDL level at baseline was 30.2±7.87 which decreasedsignificantly to 26.88±7.63 at Day 56 after treatment with CGA 7 Complexand the Percentage of VLDL levels reduced compared from baseline tovisit 4 is 10.99%.

The results collected have shown that Placebo has less improvement inlipid profile. Triglyceride level has been slightly decreased comparedfrom baseline to visit 4. The baseline triglyceride value was150.71±45.96 decreased to 149.71±45.86 at Day 56 after treatment withPlacebo and the Percentage of Triglyceride level reduced compared frombaseline to visit 4 is 0.7%.

The total cholesterol level has been slightly decreased compared frombaseline to visit 4. The baseline total cholesterol value was174.21±32.03 decreased to 173.57±31.95 at the Day 56 after treatmentwith Placebo and the Percentage of total cholesterol level reducedcompared from baseline to visit 4 is 0.4%.

The mean HDL level has shown no change compared from baseline to visit4. The baseline HDL value was 36.14±7.26 and 36.79±6.92 at Day 56 aftertreatment with Placebo.

The mean LDL slightly decreased compared from baseline to visit 4. Thebaseline LDL value was 107.93±27.03 decreased to 106.84±26.88 at Day 56after treatment with Placebo. The Percentage of LDL level reducedcompared from baseline to visit 4 is 1.0%.

The mean VLDL level has been slightly decreased compared from baselineto visit 4. The baseline VLDL value was 28.13±11.79 decreased to29.94±9.17 after treatment with Placebo and the Percentage of total VLDLlevel reduced compared from baseline to visit 4 is 6.3%.

CGA 7 Complex is statistically significant in reducing triglycerides,cholesterol, LDL and VLDL than Placebo.

CGA 7 Complex was statistically significant in increasing HDL thanplacebo. Overall, CGA 7 Complex exhibited significant reduction intriglycerides, cholesterol and LDL levels and significant increase inHDL levels.

TABLE 10 Effect of CGA7 Complex on body weight, BMI, Waist and hipcircumference, serum triglycerides, cholesterol, LDL and VLDL % S. N.Parameters Visit 1 Visit 4 Difference decrease 1 Body weight(kg) 80.6 ±10  76.57 ± 10.2 4.03 5.00 2 Body mass index(BMI kg/m²) 30.53 ± 0.6228.97 ± 0.73 1.56 5.10 3 Waist circumference(cm) 103.28 ± 4.14  99.85 ±5.36 3.43 3.32 4 Hip circumference(cm) 105.85 ± 4.88  104.78 ± 4.88 1.07 1.01 5 Triglycerides(mg/dL)   151 ± 39.39 134.43 ± 38.15 16.5710.97 6 Cholestrol(mg/dL) 172.07 ± 32.99 162.36 ± 32.10 9.71 5.64 7LDL(mg/dL) 103.96 ± 27.18  88.5 ± 29.62 15.46 14.87 8 VLDL(mg/dL)  30.2± 7.87 26.88 ± 7.63 3.32 10.99 Values are expressed in terms of Mean ±SD

TABLE 11 Effect of CGA7 Complex on serum HDL % S N. Parameters Visit 1Visit 4 Difference increase 1 HDL(mg/dL) 37.91 ± 8.29 42 ± 7.75 4.0910.78 Values are expressed in terms of Mean ± SD

TABLE 12 Effect of PLACEBO on body weight, BMI, Waist and hipcircumference, serum triglycerides, cholesterol, LDL and VLDL % S. N.Parameters Visit 1 Visit 4 Difference decrease 1 Body weight(kg) 74.03 ±9.7  73.39 ± 9.4  0.64 0.864 2 Body mass index(BMI kg/m²) 31.01 ± 1.1030.73 ± 1.18 0.29 0.90 3 Waist circumference(cm) 100.85 ± 5.89  100.57 ±4.81  0.28 0.27 4 Hip circumference(cm) 104.92 ± 7.19  104.78 ± 5.02 0.14 0.13 5 Triglycerides(mg/dL) 150.71 ± 45.96 149.71 ± 45.86 1 0.7 6Cholestrol(mg/dL) 174.21 ± 32.03 173.57 ± 31.95 0.64 0.4 7 LDL(mg/dL)107.93 ± 27.03 106.84 ± 26.88 1.1 1.01 8 VLDL(mg/dL)  28.13 ± 11.7929.94 ± 9.17 1.81 6.4 Values are expressed in terms of Mean ± SD

TABLE 13 Effect of PLACEBO on serum HDL % S.N. Parameters Visit 1 Visit4 Difference increase 1 HDL(mg/dL) 36.14 ± 7.26 36.79 ± 6.92 0.65 1.7Values are expressed in terms of Mean ± SD

Conclusion: CGA7 complex is effective in reducing body weight, reducesBMI, hip and waist circumferences, improves lipid profile by increasingHDL serum level and decreasing triglycerides, cholesterol and LDL serumlevels, and is safe with no unwanted effects.

Example 5 CGA7 Complex is Superior to Coffee Bean Extract in theManagement of Obesity, Serum Cholesterol, Triglyceride, HDL and GlucoseLevels

Obesity is a disarray of energy balance and primarily considered as adisorder of lipid metabolism. Obesity and the comorbidities associatedwith obesity remain a global health problem. It is one of the majorpublic health problems in the world because of its association with anincreased risk of various chronic diseases, including cardiovasculardiseases, type 2 diabetes, hypertension, dyslipidemia and cancers.Recent estimates in the USA indicate that approximately one-third of theadult population is obese. World Health Organization (WHO) assignsobesity as global epidemic. Globally, approximately 1.6 billion adultsare overweight and at least 400 million adults are obese. Further WHOprojects that by 2015 approximately 2.3 billion people will beoverweight and more than 700 million will be obese.

Coffee is widely used as beverages all over the world. It has numeroushealth benefits against obesity, metabolic disorders like type 2diabetes. The main active constituent of coffee is chlorogenic acidswith different isomers present in it. Chlorogenic acid is highlyantioxidant and provides protection against liver cirrhosis,atherosclerosis, bacterial infection, obesity. CGA-7 complex containsmore than 52% of seven different isomers of chlorogenic acid, extractedfrom the green coffee bean. Several in vitro and in vivo study supportthat chlorogenic acid has antiobesity activity.

With respect to this, CGA-7 complex containing more than 52% ofchlorogenic acid (Proprietary extract of Coffea arabica from VIDYA HERBSPVT LTD) was evaluated against high fat fed S.D. male rats in comparisonwith green coffee bean extract.

Objective

To investigate the role of CGA-7 Complex proprietary herbal extract ofCoffea arabica from VIDYA HERBS against high fat diet fed S.D. male ratswith compared to green coffee bean extract (GCBE).

Materials and Methods

-   -   Material: CGA 7 Complex    -   Model: High fat diet (HFD) in S.D rats    -   Composition of high fat diet: −200 g of fat/kg (170 g of lard        oil+30 g of corn oil) and 1% cholesterol.

TABLE 14 Grouping of animals Group Treatment Dose (mg/kg, orally) IControl + Vehicle Distilled water 10 ml/kg II HFD + Vehicle Distilledwater 10 ml/kg III HFD + GCBE 100 mg/kg IV HFD + CGA-7  50 mg/kg V HFD +CGA-7 100 mg/kg VI HFD + CGA-7 150 mg/kg

36 male S.D rats were divided in 6 groups each containing six. Normalrat chow diet was fed to group-1 for 42 days. HFD was fed to group 2-6for 42 days. Respective treatment was given to all groups for 42 days asdescribed in table.

Parameters Evaluated:

Body weight was measured once every 2 days. Food intake was measureddaily for 42 days.

On day 43, all the animals were kept overnight fasting before sacrifice.Blood was collected by puncturing the retro orbital plexus. Serum wasseparated by centrifugation at 3000 rpm for 10 minutes.

Serum was estimated for glucose, cholesterol, triglycerides and HDL.

Liver, mesenteric, brown adipose tissue (BAT), left and right perirenalfat pads, left and right epididymal pads were isolated and weighed.

Statistical Analysis: Data were expressed as mean±SEM & analyzed by oneway ANOVA followed by Dunnett's t test using graph pad prism version 5.Differences were considered significant at a p value of <0.05.

Results

Values are expressed in terms of SEM±Mean. Data were analyzed by one wayANOVA followed by Dunnett's t test. Number of animals in each group n=6.^(a)Comparison made with control group. ^(b)Comparison made with highfat diet group. ***P<0.001, **P<0.01

TABLE 15 Effect of CGA 7 COMPLEX on body weight in male rats Control +Vehicle 293.8 ± 1.23 145.9 ± 0.79 147.9 ± 1.09   HFD + Vehicle 360.0 ±1.21 143.6 ± 0.62  216.3 ± 1.61***^(a) HFD + GCBE (100 mg/kg) 345.7 ±5.06 144.1 ± 0.61 201.7 ± 4.49**^(b) HFD + CGA-7(50 mg/kg) 346.5 ± 3.22146.1 ± 0.50 200.3 ± 3.51**^(b) HFD + CGA-7(100 mg/kg) 311.3 ± 2.27144.1 ± 0.99  167.2 ± 3.02***^(b) HFD + CGA-7(150 mg/kg) 304.2 ± 1.81145.1 ± 1.08  159.1 ± 2.30***^(b)

TABLE 16 Effect of CGA 7 COMPLEX on average feed intake Group TreatmentFeed intake (g/per rat) I Control + Vehicle 24.05 ± 0.13 II HFD +Vehicle     17.15 ± 0.08***^(a) III HFD + GCBE (100 mg/kg)   17.88 ±0.35* ^(b) IV HFD + CGA-7(50 mg/kg) 17.48 ± 0.08 V HFD + CGA-7(100mg/kg) 17.75 ± 0.10 VI HFD + CGA-7(150 mg/kg) 17.34 ± 0.07

TABLE 17 Effect of CGA 7 COMPLEX on liver organ weight, mesenteric,brown adipose tissue (BAT), perirenal fat pads and epididymal fat padMesentric Epididymal Perirenal Liver BAT fat Fat Fat Group Treatment(per 100 g) (per 100 g) (per 100 g) (per 100 g) (per 100 g) I Control +Vehicle 2.688 ± 0.07    0.04 ± 0.002    0.57 ± 0.07    0.44 ± 0.06   0.36 ± 0.07    II HFD + Vehicle 6.467 ± 0.12***^(a)  0.15 ±0.001***^(a)  1.81 ± 0.03***^(a)  1.94 ± 0.02***^(a)  2.48 ±0.01***^(a)  III HFD + GCBE (100 mg/kg) 5.832 ± 0.17* ^(b)  0.12 ±0.002* ^(b)  1.55 ± 0.01** ^(b)  1.69 ± 0.02** ^(b)  2.09 ± 0.06** ^(b) IV HFD + CGA-7 (50 mg/kg) 5.598 ± 0.19** ^(b)  0.12 ± 0.004* ^(b)  1.54± 0.06** ^(b)  1.66 ± 0.02** ^(b)  2.07 ± 0.07** ^(b)  V HFD + CGA-7(100 mg/kg) 4.115 ± 0.19*** ^(b) 0.08 ± 0.013*** ^(b) 1.13 ± 0.08***^(b) 1.21 ± 0.09*** ^(b) 1.14 ± 0.14*** ^(b) VI HFD + CGA-7 (150 mg/kg)3.298 ± 0.17*** ^(b) 0.06 ± 0.001*** ^(b) 0.99 ± 0.01*** ^(b) 1.18 ±0.03*** ^(b) 0.98 ± 0.07*** ^(b)

Green coffee bean extract is well tolerated as anti-obesity and widelyused for same. Chlorogenic acid is chief pharmacological activeconstituents of green coffee bean. There is lack of scientific evidencesof role of chlorogenic acid in obesity management, lipid metabolism.CGA7 complex mainly contains more than 52% of seven isomers ofchlorogenic acids. The effect of CGA7 complex has been investigated inhigh fat diet induced obese S.D male rats by comparing with green coffeebean extract.

This in vivo study revealed that CGA7 complex at the dose of 100 mg/kgand 150 mg/kg has shown 25.3% and 26.4% reduction in body weight gainrespectively which are statistically significant compared to GCBEtreated group (6.7%). CGA7 complex treated groups have also loweredbrown adipose and white adipose tissue weight extensively compared toGCBE treated group.

Visceral adipose tissue discharges plenty of free fatty acids (FFA) andcytokines/hormones in the vein. Further these FFA transported to theliver where they interact with hepatocytes and various immune cells. Itis also believed that body weight and adipose tissues weight arepositively correlated with leptin and insulin levels. Cho et al studiedthat plasma leptin and insulin levels were high in HFD treated groupwhich were significantly lowered by chlorogenic acid supplementation.

Due to the leptin resistance, there is increase in FFA from adiposetissue to liver. Where FFA is converted to triglycerides and causesfatty liver by increased lipogenesis, a result of hyperinsulinemia anddecreased FFA oxidation. De sotillo and Hadley states that chlorogenicacid improves glucose tolerance and decreases plasma and hepatic lipidswithout changing triglycerides level in adipose tissue of Zucker rats at5 mg·kg (i.v.). In this study, which is conformed where HFD group hasshown increase in liver size and weight. CGA7 complex has shownsignificant reduction in liver and other adipose tissues weight whengiven orally at 100 mg/kg and 150 mg/kg compared to GCBE. CGA7 complexhas reduced serum cholesterol, triglycerides and glucose levels markedlyand improved HDL level compared to GCBE treated group.

Conclusion

CGA7 complex is superior to coffee bean extract in obesity management.CGA reduces 25.3% and 26.4% in body weight gain at 100 mg/kg and 150mg/kg respectively. CGA complex also demonstrates an ability to reduceserum cholesterol, triglyceride and glucose level, and reduce fataccumulation in liver and reduce brown and white adipose tissue. CGAcomplex improves serum HDL levels.

Example 6 Molecular Docking Studies of CGA-7 Complex with Fat Mass andObesity Associated Protein (LTO) Introduction

Weight gain and obesity are major risk factors for conditions anddiseases ranging from insulin resistance and type 2 diabetes mellitus toatherosclerosis and the sequelae of nonalcoholic fatty liver disease(Shoelson et al., 2007). Physiologically, obesity is a disarray ofenergy balance and primarily considered as a disorder of lipidmetabolism (Strader et al., 1998). The condition is associated with agrowing number of enzymes involved in lipid metabolic pathways. Theyrepresent a rich pool of potential therapeutic targets for obesity (Shi& Burn, 2004; Melnikova & Wages, 2006). Fat mass and obesity associatedprotein also known as alpha-ketoglutarate-dependent dioxygenase (FTO) isan enzyme that appears to be correlated with obesity in humans (Fraylinget al., 2007). FTO contributes to the regulation of the global metabolicrate, energy expenditure and energy homeostasis. FTO contributes to theregulation of body size and body fat accumulation.

Green coffee bean extract has been reported to have potent antiobesityand hypoglyceridemic properties by in vivo studies. Chlorogenic acidsare a major group of polyphenols found in raw green coffee bean andcontribute significantly to the pharmacological efficacy of coffee beanextract. The objective of the present study was to explore the in silicoanti-obesity activity of CGA-7 complex from green coffee bean extract bytargeting FTO.

Materials and Methods

AutoDock tools was utilized to generate grids, calculate dock score andevaluate the conformers of inhibitors bound in the active site of AMPKas targets for antidiabetic activity. Automated docking is a graphicaluser interface. AutoDock 4.2 was employed to get docking and bindingscores; which is implemented by Lamarckian genetic algorithm method. Theligand molecules i.e., the seven isomers of chlorogenic acid (FIG. 1)and Orlistat were designed and the structure was analyzed usingACD/Chemsketch. The PRODRG server was used to minimise energy of drugcompounds and 3D coordinates were prepared. The protein structure file(PDB ID: 3LFM) (FIG. 2) was taken from PDB and was edited by removingthe hetero atoms using Python molecule viewer. The grid map was centredat particular residues of the protein and was generated with AutoGrid.As per genetic algorithm all the torsions were allowed to rotate duringdocking. The Lamarckian genetic algorithm and the pseudo-Solis and Wetsmethods were applied for minimization, using default parameters(Rodriguez and Infante, 2011).

Results

All the seven isoforms of chlorogenic acid in CGA-7 binds veryefficiently within the active pocket of FTO (FIG. 3). The resultobtained is comparable to orientation of standard drug Orlistat. Thebinding energy required is less than that of Orlistat and the firmbinding of isoforms is evident from the formation of more hydrogen bondsof ligand molecules in comparison with standard drug (Table 1).

TABLE 18 Molecular docking results of Fat mass and obesity associatedprotein Thermodynamic parameters Binding Ligand Molecule energyefficiency H-bonds Interactions 3-O-Caffeoylquinic −8.04 −0.32 1.28−11.32 5 Leu78 acid His73 Arg80 Gln468 4-O-Caffeoylquinic −7.01 −0.287.21 −10.3 7 Leu78 acid Lys391 Asp467 Arg80 5-O-Caffeoylquinic −7.43−0.3 3.56 −10.7 6 Arg80 acid Leu78 His73 Arg80 Gln468 5-O-Feruloylquinic−5.56 −0.21 84.65 −8.84 5 His73 acid Arg80 Gln468 Leu783,4-O-Dicaffeoylquinic −5.32 −0.21 126.75 −8.6 4 His73 acid Gln468 Arg803,5-O-Dicaffeoylquinic −5.88 −0.16 48.78 −10.95 6 Asp208 acid His73Gln468 Lys74 Lys391 4,5-O-Dicaffeoylquinic −7.73 −0.26 2.14 −12.81 5Arg80 acid Leu78 Lys391 His73 Orlistat (Std) −4.65 −0.14 390.87 −11.21 3Ser95 Arg80

Conclusion

CGA-7 as a complex mixture of 7 isoforms of chlorogenic acid from greencoffee bean extract is effective in interacting with fat mass andobesity associated protein (FTO) more efficiently than the standard drugOrlistat and hence may serve as a better candidate for the developmentof anti-obesity drugs in future.

The embodiments herein and the various features and advantageous detailsthereof are explained with reference to the non-limiting embodiments inthe description. Descriptions of well-known components and processingtechniques are omitted so as to not unnecessarily obscure theembodiments herein. The examples used herein are intended merely tofacilitate an understanding of ways in which the embodiments herein maybe practiced and to further enable those of skill in the art to practicethe embodiments herein. Accordingly, the examples should not beconstrued as limiting the scope of the embodiments herein.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the embodiments herein that others can, byapplying current knowledge, readily modify and/or adapt for variousapplications such specific embodiments without departing from thegeneric concept, and, therefore, such adaptations and modificationsshould and are intended to be comprehended within the meaning and rangeof equivalents of the disclosed embodiments. It is to be understood thatthe phraseology or terminology employed herein is for the purpose ofdescription and not of limitation. Therefore, while the embodimentsherein have been described in terms of preferred embodiments, thoseskilled in the art will recognize that the embodiments herein can bepracticed with modification within the spirit and scope of theembodiments as described herein.

Any discussion of documents, acts, materials, devices, articles and thelike that has been included in this specification is solely for thepurpose of providing a context for the disclosure. It is not to be takenas an admission that any or all of these matters form a part of theprior art base or were common general knowledge in the field relevant tothe disclosure as it existed anywhere before the priority date of thisapplication.

While considerable emphasis has been placed herein on the particularfeatures of this disclosure, it will be appreciated that variousmodifications can be made, and that many changes can be made in thepreferred embodiments without departing from the principles of thedisclosure. These and other modifications in the nature of thedisclosure or the preferred embodiments will be apparent to thoseskilled in the art from the disclosure herein, whereby it is to bedistinctly understood that the foregoing descriptive matter is to beinterpreted merely as illustrative of the disclosure and not as alimitation.

The disclosures of all references mentioned in this application areincorporated by reference in their entirety as if set forth verbatimherein.

REFERENCES

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We claim:
 1. A composition of comprising: about 7.23% 3-caffeoylquinicacid (3-CQA), about 25.43% 5-caffeoylquinic acid (5-CQA), about 9.51%4-caffeoylquinic acid (4-CQA), about 1.84% 5-feruloylquinic acid(5-FQA), about 2.79% 3,4 dicaffeoylquinic acid (3,4 di CQA), about 1.90%3,5 dicaffeoylquinic acid (3,5 di CQA), and about 3.74% 4,5dicaffeoylquinic acid (4,5 di CQA).
 2. The composition of claim 1,wherein the composition is formulated as a pill, tablet, capsule, bead,lozenge, gummy, gel, liquid, chew, powder, soluble strip or foam.
 3. Amethod for treating obesity in a subject comprising administering to thesubject the composition of claim
 1. 4. A method for making a compositionfor the treatment of obesity comprising: providing green coffee beans;subjecting the green coffee beans to extraction in a solvent at 75-78°C. for about 7 to 8 hours to produce a bed of green coffee beans and anextract; subjecting the bed of green coffee beans to a solvent at 75-78°C. for about 7 to 8 hours three times to produce a bed extract;filtering the extract and the bed extract to produce a filtered extractand concentrating the filtered extract by removing the solvent toproduce a green coffee complex; dissolving the green coffee complex in asolvent and subjecting the dissolved green coffee complex to resinextraction thereby collecting a purified green coffee complex on theresin; eluting the purified green coffee extract from the resin with asolvent to produce a soluble pure green coffee complex; and producing aconcentrated pure green coffee complex by removing the solvent from thesoluble pure green coffee complex; spray drying the concentrated solublepure green coffee complex to produce a dried chlorogenic acidcomposition.
 5. The method of claim 3, wherein the chlorogenic acidcomposition is suitable for treating obesity in a subject.
 6. The methodof claim 3, wherein the chlorogenic acid composition is formulated as apowder, pill, tablet, pellet, capsule, thin film, solution, spray,syrup, linctus, lozenge, pastille, chewing gum, paste, vapor,suspension, emulsion, ointment, cream, lotion, liniment, gel, drop,topical patche, buccal patch, bead, gummy, gel, sol or injection.